Nanopesticide risk assessment based on microbiome profiling - Community structure and functional potential as biomarkers in captan@ZnO35-45 nm and captan@SiO220-30 nm treated orchard soil.

Nanoformulation should minimise the usage of pesticides and limit their environmental footprint. The risk assessment of two nanopesticides with fungicide captan as an active organic substance and ZnO35-45 nm or SiO220-30 nm as nanocarriers was evaluated using the non-target soil microorganisms as biomarkers. The first time for that kind of nanopesticides next-generation sequencing (NGS) of bacterial 16 S rRNA and fungal ITS region and metagenomics functional predictions (PICRUST2) was made to study structural and functional biodiversity. During a 100-day microcosm study in soil with pesticide application history, the effect of nanopesticides was compared to pure captan and both nanocarriers. Nanoagrochemicals affected microbial composition, especially Acidobacteria-6 class, and alpha diversity, but the observed effect was generally more substantial for pure captan. As for beta diversity, the negative impact was detected only in response to captan and still observed on day 100. Fungal community in the orchard soil showed only a decrease in phylogenetic diversity in captan set-up since day 30. PICRUST2 analysis confirmed several times lower impact of nanopesticides considering the abundance of functional pathways and genes encoding enzymes. Furthermore, the overall data indicated that using SiO220-30 nm as a nanocarrier speeds up a recovery process compared to ZnO35-45 nm.

Copper-oxide nanoparticles exert persistent changes in the structural and functional microbial diversity: A 60-day mesocosm study of zinc-oxide and copper-oxide nanoparticles in the soil-microorganism-nanoparticle system.

Recent advances in nanotechnology and development of nanoformulation methods, has enabled the emergence of precision farming - a novel farming method that involves nanopesticides and nanoferilizers. Zinc-oxide nanoparticles serve as a Zn source for plants, but they are also used as nanocarriers for other agents, whereas copper-oxide nanoparticles possess antifungal activity, but in some cases may also serve as a micronutrient providing Cu ions. Excessive application of metal-containing agents leads to their accumulation in soil, where they pose a threat to non-target soil organisms. In this study, soils obtained from the environment were amended with commercial zinc-oxide nanoparticles: Zn-OxNPs(10-30), and newly-synthesized copper-oxide nanoparticles: Cu-OxNPs(1-10). Nanoparticles (NPs) in 100 and 1000 mg kg-1 concentrations were added in separate set-ups, representing a soil-microorganism-nanoparticle system in a 60-day laboratory mesocosm experiment. To track environmental footprint of NPs on soil microorganisms, a Phospholipd Fatty Acid biomarker analysis was employed to study microbial community structure, whereas Community-Level Physiological Profiles of bacterial and fungal fractions were measured with Biolog Eco and FF microplates, respectively. The results revealed a prominent and persistent effects exerted by copper-containing nanoparticles on non-target microbial communities. A severe loss of Gram-positive bacteria was observed in conjunction with disturbances in bacterial and fungal CLPPs. These effects persisted till the end of a 60-day experiment, demonstrating detrimental rearrangements in microbial community structure and functions. The effects imposed by zinc-oxide NPs were less pronounced. As persistent changes were observed for newly synthesized Cu-containing NPs, this work stresses the need for obligatory testing of nanoparticle interactions with non-target microbial communities in long-term experiments, especially during the approval procedures of novel nano-substances. It also underlines the role of in-depth physical and chemical studies of NP-containing agents, which may be tweaked to mitigate the unwanted behavior of such substances in the environment and preselect their beneficial characteristics.

The influence of ZnO nanoparticles on horizontal transfer of resistance genes in lab and soil conditions.

Antibiotic resistance is a severe problem that threatens the achievements of modern medicine. Metallic nanoparticles may promote the horizontal transfer of resistance genes due to their toxicity to bacterial cells and metal-induced co-selection mechanisms. In this study, we investigated the toxicity of ZnO nanoparticles to E. coli DH5α laboratory strain and the abundance of soil microbial community. Moreover, the influence of ZnO nanoparticles on resistance gene transfer in laboratory and soil conditions was evaluated. ZnO nanoparticles at concentrations up to 10 mg L-1 reduced the survival of E. coli cells by 14.6% and increased the transformation frequency by almost 1.8 fold. In soil, ZnO nanoparticles at a concentration of 1000 mg kg-1 affected the total abundance of bacteria, causing a decrease in the 16S rRNA gene copy number. We did not detect the presence of 11 target antibiotic resistance genes (sul1, sul2, imp2, imp5, blaCTX-M, ermB, mefA, strB, aadA1, tetA1, tetB), which confer resistance to five classes of antibiotics in soil treated with ZnO nanoparticles. No elevated conjugation frequency was observed in soil microbial communities treated with ZnO nanoparticles. However, the increase in czcA gene copies indicates the spread of genetic elements harbouring metal resistance. The data shows that metallic nanoparticles promote the spread of antibiotic and metal resistance genes. The broad implication of the present research is that the inevitable nanoparticles environmental pollution may lead to the further dissemination of antibiotic resistance and profoundly influence public health.

Negligible Effect of Estrogen Deficiency on Development of Skeletal Changes Induced by Type 1 Diabetes in Experimental Rat Models.

Although postmenopausal osteoporosis often occurs concurrently with diabetes, little is known about interactions between estrogen deficiency and hyperglycemia in the skeletal system. In the present study, the effects of estrogen deficiency on the development of biochemical, microstructural, and mechanical changes induced by streptozotocin-induced diabetes mellitus (DM) in the rat skeletal system were investigated. The experiments were carried out on nonovariectomized (NOVX) and ovariectomized (OVX) control and diabetic mature female Wistar rats. Serum levels of bone turnover markers (CTX-I and osteocalcin) and 23 cytokines, bone mass and mineralization, histomorphometric parameters, and mechanical properties of cancellous and compact bone were determined. The results were subjected to two-way ANOVA and principal component analysis (PCA). Estrogen deficiency induced osteoporotic changes, with increased bone resorption and formation, and worsening of microstructure (femoral metaphyseal BV/TV decreased by 13.0%) and mechanical properties of cancellous bone (the maximum load in the proximal tibial metaphysis decreased by 34.2%). DM in both the NOVX and OVX rats decreased bone mass, increased bone resorption and decreased bone formation, and worsened cancellous bone microarchitecture (for example, the femoral metaphyseal BV/TV decreased by 17.3% and 18.1%, respectively, in relation to the NOVX controls) and strength (the maximum load in the proximal tibial metaphysis decreased by 35.4% and 48.1%, respectively, in relation to the NOVX controls). Only in the diabetic rats, profound increases in some cytokine levels were noted. In conclusion, the changes induced by DM in female rats were only slightly intensified by estrogen deficiency. Despite similar effects on bone microstructure and strength, the influence of DM on the skeletal system was based on more profound systemic homeostasis changes than those induced by estrogen deficiency.

Chrysin Reduces Oxidative Stress but Does Not Affect Polyol Pathway in the Lenses of Type 1 Diabetic Rats.

Prolonged hyperglycemia is one of the main causes of reactive oxygen species and free radicals generation in diabetes which may affect various organs, including the eye. Oxidative damage to proteins and lipids in the eye lens could lead to cataract formation. To cope with oxidative stress, the endogenous antioxidative system may be supported by the supplementation of exogenous antioxidants. The aim of this study was to evaluate the effect of chrysin, a natural flavonoid, on oxidative stress and polyol pathway-related markers in the lenses of streptozotocin-induced type 1 male diabetic rats. Chrysin at doses of 50 and 100 mg/kg was administered by gavage for 28 days. This treatment resulted in a decrease in antioxidative enzymes activity and oxidative stress index. Moreover, chrysin administration elevated the reduced glutathione level in the lenses. A decrease in the markers linked to oxidative damage to proteins and lipids in the lenses was noted, especially after treatment with 50 mg/kg of chrysin. Neither of the chrysin doses affected glycemia-related markers in the serum or altered parameters related to the polyol pathway and advanced glycation end-products level in the lenses of diabetic rats. Upon obtaining results, it can be concluded that chrysin reveals antioxidative activity in the lenses but shows no antihyperglycemic or antiglycation properties.

Effect of Rosmarinic Acid and Sinapic Acid on Oxidative Stress Parameters in the Cardiac Tissue and Serum of Type 2 Diabetic Female Rats.

Cardiovascular diseases are one of the most common complications of type 2 diabetes. They are considered the leading cause of death among diabetics. One of the mechanisms underlying diabetic cardiovascular complications is oxidative stress. Many phenolic acids are regarded as antioxidants. The aim of the study was to investigate the effect of rosmarinic acid (RA) and sinapic acid (SA) on oxidative stress parameters in the cardiac tissue and serum of type 2 diabetic female rats. Additionally, the effect of these compounds on glucose homeostasis and lipid profile in the serum was evaluated. Type 2 diabetes was induced with high-fat diet and streptozotocin. RA at the doses of 10 and 50 mg/kg and SA at the doses of 5 and 25 mg/kg were administrated orally for 28 days. Untreated diabetic rats exhibited unfavorable changes in glucose metabolism and lipid profile. Changes in the enzymatic and non-enzymatic markers indicated the onset of oxidative stress in these animals. The results showed that the higher doses of the tested phenolic acids-50 mg/kg of RA and 25 mg/kg of SA-revealed beneficial effects on oxidative stress in the cardiac tissue of diabetic rats.

Plant Species and Heavy Metals Affect Biodiversity of Microbial Communities Associated With Metal-Tolerant Plants in Metalliferous Soils.

We here assess the biodiversity of the rhizosphere microbial communities of metal-tolerant plant species Arabidopsis arenosa, Arabidopsis halleri, Deschampsia caespitosa, and Silene vulgaris when growing on various heavy metal polluted sites. Our broad-spectrum analyses included counts for total and metal-tolerant culturable bacteria, assessments of microbial community structure by phospholipid fatty acid (PLFA) profiling and community-level analysis based on BIOLOG-CLPP to indicate functional diversity. The genetic-biochemical diversity was also measured by denaturing gradient gel electrophoresis (PCR-DGGE) and metabolomic analysis (HPLC-MS). Different rhizospheres showed distinctive profiles of microbial traits, which also differed significantly from bulk soil, indicating an influence from sampling site as well as plant species. However, total bacterial counts and PCR-DGGE profiles were most affected by the plants, whereas sampling site-connected variability was predominant for the PLFA profiles and an interaction of both factors for BIOLOG-CLPP. Correlations were also observed between pH, total and bioavailable Cd or Zn and measured microbial traits. Thus, both plant species and heavy-metals were shown to be major determinants of microbial community structure and function.

Variable Effects of Non-steroidal Anti-inflammatory Drugs (NSAIDs) on Selected Biochemical Processes Mediated by Soil Microorganisms.

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most frequently used group of pharmaceuticals. The high consumption and the uncontrolled disposal of unused drugs into municipal waste or their deposit in landfills can result in an increased concentration of these compounds in soils. Moreover, these drugs can affect the microbial activity. However, there is a lack of knowledge about these effects or it is very limited. Therefore, the objective of this study was to compare the impact of selected commercially available NSAIDs, i.e., diclofenac (DCF), naproxen (NPX), ibuprofen (IBF) and ketoprofen (KTP), applied at concentrations of 1 and 10 mg/kg soil, on the activity of soil microorganisms during the 90-day experiment. To ascertain this impact, substrate-induced respiration (SIR), soil enzyme activities, i.e., dehydrogenase (DHA), acid and alkaline phosphatases (PHOS-H and PHOS-OH) and urease (URE) as well as changes in the rates of nitrification and ammonification processes were determined. In addition, the number of culturable bacteria and fungi were enumerated. In general, the obtained data showed a significant stimulatory effect of NSAIDs on the microbial activity. Higher concentrations of NSAIDs caused a greater effect, which was observed for SIR, PHOS-H, PHOS-OH, URE, N-NO3- and N-NH4+, even during the whole incubation period. Moreover, the number of heterotrophic bacteria and fungi increased significantly during the experiment, which was probably a consequence of the evolution of specific microorganisms that were capable of degrading NSAIDs and used them as an additional source of carbon and energy. However, an inhibitory effect of NPX, IBF or KTP for SIR, DHA, on both phosphatases and culturable bacteria and fungi was observed at the beginning of the experiment. At lower concentrations of NSAIDs, in turn, the effects were negligible or transient. In conclusion, the application of NSAIDs altered the biochemical and microbial activity of soil what may cause the disturbance in soil functioning. It is reasonable to assume that some components of the NSAID formulations could stimulate soil microorganisms, thus resulting in an increase in biochemical activities of the soil.

An Analysis of the Effects of Vancomycin and/or Vancomycin-Resistant Citrobacter freundii Exposure on the Microbial Community Structure in Soil.

The occurrence of antibiotics and antibiotic resistance genes in the environment has become a subject of growing concern. The extensive use of vancomycin and other pharmaceuticals may alter the biodiversity of soil microbial communities and select antibiotic-resistant bacteria. Therefore, the purpose of the study was to evaluate the impact of vancomycin and/or vancomycin-resistant Citrobacter freundii on soil microbial communities using the denaturing gradient gel electrophoresis (DGGE) and the phospholipid fatty acid (PLFA) approaches. The experiment had a completely randomized block design with the following treatments: control soil (C), soil with vancomycin (1 mg/kg soil-VA1), soil with vancomycin (10 mg/kg soil-VA10), soil with C. freundii (Cit), soil with vancomycin (1 mg/kg soil) and C. freundii (VA1+Cit), and soil with vancomycin (10 mg/kg soil) and C. freundii (VA10+Cit). A bacterial strain resistant to vancomycin was isolated from raw sewage collected from the municipal sewage treatment plant. The obtained results indicated that the antibiotic and/or the bacterial strain exerted a selective pressure that resulted in qualitative and quantitative changes in the population of soil microorganisms. However, a multivariate analysis showed that the genetic and structural diversity of the soil microbial community was primarily affected by the incubation time and to a lesser extent by the antibiotic and introduced bacteria. DGGE analysis clearly showed that certain species within the bacterial community were sensitive to vancomycin as was evidenced by a decrease in the values of S (richness) and H (Shannon-Wiener) indices. Moreover, a PLFA method-based analysis revealed alterations in the structure of the soil microbial community as indicated by changes in the biomass of the PLFA biomarkers specific for Gram-positive and Gram-negative bacteria as well as fungi. The changes observed in the community of soil microorganisms may decrease the rate of microbial-mediated processes, which can lead to a disturbance in the ecological balance of the soil ecosystem.

Imidacloprid induces changes in the structure, genetic diversity and catabolic activity of soil microbial communities.

This is the first report describing the effect of imidacloprid applied at field rate (FR, 1 mg/kg of soil) and 10 times the FR (10*FR, 10 mg/kg of soil) on the structural, genetic and physiological diversity of soil bacterial community as determined by the phospholipid fatty acid (PLFA), the denaturing gradient gel electrophoresis (DGGE), and the community level physiological profile (CLPP) approaches. PLFA profiles showed that imidacloprid significantly shifted the microbial community structure and decreased the biomass of the total, bacterial and fungal PLFAs, however, this effect was transient at the FR dosage. The alterations in DGGE patterns caused by imidacloprid application, confirmed considerable changes in the overall richness and diversity of dominant bacteria. Although, as a result of imidacloprid application, the metabolic activity of microbial communities was generally lower, the richness and functional biodiversity of the soil microbial community were not negatively affected. In general, the analysis of the variance indicated that the measured parameters were significantly affected by treatment and the incubation time, however, the incubation time effect explained most of the observed variance. Imidacloprid degradation and the appearance of some new bands in DGGE profiles suggest the evolution of bacteria capable of degrading imidacloprid among indigenous microflora.

A broad-spectrum analysis of the effects of teflubenzuron exposure on the biochemical activities and microbial community structure of soil.

We evaluated the response of soil bacteria to applications of the insecticide teflubenzuron at the field rate dosage (FR; 0.15 mg/kg of soil) and at a higher dosage (10*FR; 1.5 mg/kg of soil). When applied at the FR dosage, teflubenzuron had no effect on several biochemical parameters of the soil, including substrate-induced respiration (SIR), dehydrogenase (DHA) and phosphatase activities (PHOS), and N-NO(3)(-) and N-NH(4)(+) concentrations. Additionally, no differences were observed in the culturable fraction of the soil bacteria (the number of heterotrophic, nitrifying, denitrifying and N(2)-fixing bacteria; the growth strategy; the ecophysiological and colony development indices; and the physiological state). In contrast, treatment with the 10*FR dosage of the insecticide significantly increased SIR, DHA, PHOS and N-NH(4)(+) levels and the number of heterotrophic and denitrifying bacteria. Decreases in urease activity (URE) and the number of nitrifying and N(2)-fixing bacteria were also observed. A phospholipid fatty acid (PLFA) method-based analysis of the entire soil microorganism population revealed that teflubenzuron treatment affected the total fatty acid level as well as those considered to be of Gram-positive bacteria, Gram-negative bacteria and fungi. This effect was observed on days 1 and 14 post-treatment. A principal component analysis (PCA) of the PLFAs showed that teflubenzuron treatment significantly shifted the microbial community structure; however, all of the observed effects were transient. Studies on the degradation of teflubenzuron revealed that this process is characterised by a short lag phase and a rate constant (k) of 0.020/day. This degradation rate follows first-order kinetics, and the DT50 was 33.5 days. This is the first study that thoroughly examines the functional and structural status of both the culturable and non-culturable fractions of the soil microbial community after teflubenzuron application.